When the adenosine triphosphate (ATP) pool in a cell is depleted below the level which must be maintained to meet the cellular needs for maintenance of metabolic processes, the cell is not only incapable of mitotic division but the cell dies. The rate of change in the ATP pool size existing in a cell at any particular time is the difference between the rate at which ATP is being produced, primarily by oxidative phosphorylation along the respiratory chain (RC) in the mitochondria, and the rate at which ATP is being used up (hydrolyzed) to provide substantially all the energy requirements of the cell. This energy is principally required for all the myriad anabolic and catabolic reactions in the metabolism of the cell, and for powering the "sodium pumps" of the pericellular membrane--whose collective action keeps the intracellular Na.sup.+ -concentration relatively low despite the continuous leakage of Na.sup.+ through the membrane into the cell from the high Na.sup.+ -concentration extracellular fluid. The fundamental pathway involved in ATP production and usage (hydrolysis) in all normal body cells is depicted in FIG. 1.
The abbreviations used in FIG. 1 and elsewhere throughout this application are explained in the following table:
TABLE ______________________________________ AA amino acids AcCoA acetyl coenzyme A ADP adenosine diphosphate Amr active metabolic rate ATP adenosine triphosphate, the basic compound for storing chemical energy in the cell ATPase adenosine triphosphatase Bmr basal metabolic rate (expressed as a multiple of pretreatment Bmr or Mayo Normal Standard Bmr) Ca calcium CAC Citric Acid Cycle Cho carbohydrate component of Dnr Cl.sup.- ion d day DNP 2,4-dinitrophenol Dnr defined nutritional regimen Efa essential fatty acid component of Dnr EMP Embden-Meyerhof Pathway Emr.sub.A effective (average) metabolic rate FA fatty acids g gram I iodine Kcal kilocalories kg kilogram lO.sub.2 /d liters of O.sub.2 consumed metabolically, per day (24 hours) Mg magnesium mg milligram ml milliliter Mn manganese Na.sup.+ sodium ion NADH reduced nicotinamide adenine dinucleotide O.sub.2 molecular oxygen O/P oxidative phosphorylation P phosphorus Pr protein component of Dnr Pr = 15 denotes protein allowance basis for Dnr-protein; 15 g protein per 70 kg body weight Pr.sub.min minimum protein allowance to maintain nitrogen equilibrium RC respiratory chain Se selenium SP sodium pump UA uncoupling agent V + M vitamins + minerals mix (daily amount supplied) W.sub.B body weight (kg) Zn zinc ______________________________________
In normal (i.e., nonmalignant) body cells, the key nutritional component from which the fundamental energy supply for synthesizing ATP is derived is glucose. Glucose is transformed by the sequential reactions of the Glycolytic or Embden-Meyerhof Pathway (EMP) into pyruvate. Subsequently, pyruvate is decarboxylated and forms acetyl coenzyme A (AcCoA) which then enters the citric acid cycle (CAC) in the mitochondria. Here each acetate moiety, after first being incorporated into a molecule of citric acid, is broken down into CO.sub.2 and H with the H appearing, inter alia, in molecules of reduced nicotinamide adenine dinucleotide (NADH) which then contain a large fraction of the energy contained in the original glucose. This NADH subsequently is oxidized in the mitochondrial respiratory chain (RC) with the ultimate production of H.sub.2 O by terminal reaction of the H with molecular O.sub.2 ; this O.sub.2 is readily supplied by the normal vasculature. The energy obtained by the transport of electrons down the potential gradient of the RC, by a sequence of redox reactions, is used to produce the ATP of the cell. Thus, in normal cells, the ATP-stored energy is obtained in the major proportion from nutritional glucose or from carbohydrates (i.e., starches and sugars) yielding glucose upon digestion. Some ATP-energy is obtained in normal cells from the oxidation, in the citric acid cycle, of fatty acids and amino acids obtained from nutritional fats and proteins. When adequate glucose is available in the nutriment intake, however, all major ATP-energy needs of normal cells are readily obtainable from glucose alone. The ATP produced in the respiratory chain enters the cellular "ATP Pool", from which it is continuously withdrawn to supply the energy needs of total cellular metabolism and to power the membrane sodium pumps which keep the intracellular Na.sup.+ -concentration adequately low by the outpumping of Na.sup.+.
This same general pattern of ATP generation and usage obtains in malignant cells, but with two crucial differences. First, it has been extensively demonstrated that malignant cells in general possess a distinctive metabolic aberrancy, ostensibly as an innate consequence of their transformation to the malignant state. Under in vivo conditions, malignant cells in tumors do not substantially convert pyruvate to AcCoA (see FIG. 2); the pyruvate instead is essentially converted to lactate and is excreted from the cell. [Busch, H., An Introduction to the Biochemistry of the Cancer Cell Chapter 10, Academic Press, New York (1962)]. The net consequence is that only a very small fraction of the chemical energy in glucose can be extracted and used by the cancer cell, compared to that available to the normal cell. Since nutritional glucose is by far the most prominent and important source of normal cellular ATP energy under normal conditions, this transformation aberrancy puts the malignant cell at a great disadvantage regarding the maximal rates at which it can generate ATP. This metabolic defect is particularly detrimental for the malignant cell, which generally needs an especially abundant supply rate of ATP to support the highly active metabolism associated with the frequent mitosis characteristic of these highly proliferative cells.
However, the malignant cell quite effectively circumvents this deficiency under usual nutritional conditions by ready oxidation of fatty acids and amino acids in the citric acid cycle. Mitochondria possess a very efficient enzyme system capable of effecting the ".beta.-oxidization" of fatty acids directly to AcCoA, which then enters the citric acid cycle and is oxidized exactly as the AcCoA produced from glucose is oxidized in normal cells. The amino acids are similarly reduced to AcCoA or other intermediates of the CAC and then oxidized, after initial deamination. Thus, some amino acid species are capable of entering the citric acid cycle directly at various intermediate points of the cycle, after deamination and suitable transformation, all readily accomplished by the enzyme systems of the malignant cell. Consequently, although substantially deprived of the utilization of glucose as a primary energy source, the malignant cell makes full use of the supply of the energy-rich fatty acids, and amino acids, all present in the plasma under usual nutritional intake level. In anorexic patients having low food intake in the very late stages of malignancy, the profound cachexia observed attests to the effectiveness with which fat and protein (muscle) depots have been mobilized, and thus fatty acids and amino acids made available to the malignant cells for their continued proliferation, while the patient becomes emaciated.
In accordance with the present invention, the ATP pool of malignant cells in the body is depleted to a level which is inadequate for maintenance of the essential metabolic processes of these cells, without substantially altering the normal ATP pool size in the normal cells of the body.
The present therapy consists of two parts, administered concurrently. The first part of the therapy is designed to severely limit the maximum rate at which the cancer cells can potentially produce ATP via the respiratory chain (RC), without limiting to any significant extent the rate at which normal cells can potentially produce ATP. The second part is designed to grossly reduce the actual net ATP production rate of the cancer cells by uncoupling a major part of their oxidative phosphorylation, without altering the actual ATP production rate of the normal cells from their normal level. The pronounced net deficit in the ATP production rate, relative to that necessary just to supply the minimal ATP rate requirements of the essential metabolic processes, soon reduces the ATP pool size selectively in the cancer cells to a subminimal level inadequate for continued vital functioning. Degeneration, lysis, or functional death of the cancer cells then ensues.
The first part of the therapy system comprises the administration of a defined nutritional regimen (Dnr) which consists essentially of a nutritional regimen designed to maximize the use of nutritional carbohydrates as a source of ATP energy, and to minimize the use of nutritional fatty acids and amino acids for the same purpose.
The second part comprises a concurrently administered dosage of an agent effective to uncouple oxidative phosphorylation (UA) so as to greatly reduce the net ATP production rate of the cancer cells by uncoupling a large fraction of the maximum potential ATP production per unit time, a maximum already severely limited by the reduced availability of NADH resulting from the restriction of available fatty acids and amino acids by the Dnr. Since the normal cells can make full use of the abundant carbohydrate (glucose) supplied by the Dnr for energy purposes, the only effect on the normal cells is an increase in O.sub.2 consumption rate; the potential ATP loss in the normal cells due to the uncoupling action is fully compensated by a higher rate of NADH oxidation by the respiratory chain, while the rate of actual net ATP production remains unchanged at its usual, normal level.
This invention encompasses the novel use, as effective anticancer agents in vivo in humans, of physiologically tolerable agents which uncouple oxidative phosphorylation. Applicant has demonstrated that the classical uncoupler 2,4-dinitrophenol (DNP), when used with the nutritional regimen of this invention, will bring about a rapid and marked reduction of size in a variety of malignant tumor types in humans. Such size reduction is characteristic clinical evidence for malignant cell lysis and degeneration, also termed oncolysis. Applicant's test results and other available information indicate that a like reduction in malignant cell content of tissues containing disperse or otherwise nonaggregated malignant cells will result from treatment in accordance with this invention.
Applicant has disclosed a related nutritional regimen as part of a different system for the treatment of cancer in U.S. patent application Ser. No 223,850, filed Jan. 9, 1981, but did not disclose the use of DNP or of other physiologically tolerable uncoupling agents as anticancer agents.
Physiologically tolerable agents that uncouple the oxidative energy-releasing centers of the respiratory chain from the ATP-yielding phosphorylation of adenosine diphosphate in the mitochondria of cells have been investigated extensively over the past thirty-five years in the study of oxidative metabolism [Demers, L. M. et al. Proc, Soc. Exper. Med. 140, 724 (1972); Hemker, H. C. Biochem. Biophys. Acta 63, 46 (1962); Hemker, H. C. Biochem. Biophys. Acta 48, 221 (1961); Heytler, P. G. "Uncouplers of Oxidative Phosphorylation" in Erecinski et al (eds.) Inhibitors of Mitochondrial Functions Pergamon 1981, p. 203]. Indeed it was observations of the effects of such agents on cell respiration that led to the discovery of the fundamental process of oxidative phosphorylation [Hotchkiss, Adv. Enzymol. 4, 153 (1944)]. Because of the vital importance of aerobic metabolism as the major source of cellular ATP production, even physiologically tolerable agents capable of effecting a substantial degree of uncoupling of oxidative phosporylation are potentially very toxic in excessive dosage and must obviously be utilized with great care. The underlying basis of all toxic effects in normal cells due to excessive uncoupling of oxidative phosphorylation lies in the concomitant reduction of the cellular ATP production rate below that required to support the essential metabolic needs of the cell for normal functioning. Consequently, since the principal effect of uncoupling is an accelerated rate of oxidation of NADH by the RC with a commensurate elevation in the O.sub.2 consumption rate, the relative level of uncoupling by an uncoupling agent (UA) can be directly monitored by measurement of the whole-body basal metabolic rate (Bmr), in terms of lO.sub.2 /d. Thus, safe UA dosage ranges can be simply and effectively determined by careful monitoring of the Bmr, in conjunction with careful monitoring of carbohydrate intake to insure it meets the total daily caloric needs (Emr.sub.A) of the body.
In the first major medical use of an uncoupling agent, the use of DNP for reduction of obesity, the absolute need for monitoring the Bmr to insure the maintenance of a proper level of safety was pointed out by the original investigators [Tainter, M. L. et al. J. Am. Med. Assoc. 101, 1472 (1933); Tainter, M. L. et al. J. Phramacol. Exp. Therap. 48, 410 (1933)], who emphasized that the administration of DNP must be performed only under close medical supervision and monitoring. These and other clinical investigators conducted a preliminary investigation of the dosage-effects properties of DNP in a wide range of animals and humans, and demonstrated the essential nonexistence of any deleterious side effects of DNP when the Bmr was held at the desired clinical level by administration of the appropriate dosage level of DNP [Borley, W. E. et al. Arch. Opth. 18, 908 (1937); Borley, W. E. et al. Am. J. Ophth. 21, 1091 (1938); Cutting, W. C. et al J.A.M.A. 101, 193 (1933); Cutting, W. C. et al. J. Clin. Investigation 13, 547 (1934); Schulte, T. L. J. Pharm. Exper. Biol. Med. 419 (1937); Schulte, T. L. et al. Proc. Soc. Exper. Ther. 31, 1163 (1934); Tainter, M. L. J. Pharm. Exper. Ther. 49, 187 (1933); Tainter, M. L. J. Pharm. Exper. Ther 51, 143 (1934); Tainter, M. L. Proc. Soc. Exper Biol Med. 31, 1161 (1934); Tainter, M. L. J.A.M.A. 104, 1071 (1935); Tainter, M. L. J. Pharm. Exper Ther. 63,51 (1938); Tainter, M. L. et al. J. Pharm. Exper. Ther. 53, 58 (1935); Tainter, M. L. et al. Arch. Ophth 29, 30 (1938); Tainter, M. L. et al. J. Pharm. Exper. Ther. 55, 326 (1935); Tainter, M. L. et al. Am. J. Pub. Health 24, 1045 (1934); Tainter, M. L. et al. Arch. Path. 18, 881 (1934); Tainter, M. L. et al. J.A.M.A. 101, 1472 (1933); Tainter, M. L. et al. J.A.M.A. 102, 1147 (1934); Terada, B. et al. J. Pharm. Exper. Ther. 54, 454 (1935)].
Unfortunately, the early success in the clinical use of DNP for obesity reduction soon led to its wide and indiscriminate use by the public, without professional supervision, for weight reduction. A multitude of weight reduction nostrums containing unspecified concentrations of DNP appeared on the non-prescription market. Abuse and overdoses, some even for suicidal purposes, yielded a complete profile on human toxicity effects [Parascondola, J. L. Molecular and Cellular Biochemistry 5, 69 (1974)]. A chronic toxic effect possibly related to DNP observed among the population at large in individuals on uncontrolled and unsupervised weight reduction programs, was the formation of cataracts in a small number of cases. [Horner, D. W. Arch. Opth. 27, 1097 (1942)]. However, such cataract formation, at much higher incidence levels, is particularly common in a number of physiological conditions in which there is hypoglycemia or an inability to transport glucose into cells at an adequate rate (starvation, chronic hypoglycemia, and diabetes mellitus), and the observed cataracts may have been the result of weight-reduction-associated hypoglycemia rather than a direct effect of DNP itself. The therapy system of the present invention innately and effectively insures the maintenance of normal or higher blood glucose levels at all times. Cataract formation has not been observed in any of a wide range of animal species given DNP, even at high dosages [Horner, D. W. supra]. Hitch, J. M. et al., J. Am. Med. Assn. 106, 2130 (1936) suggests a relationship between DNP ingestion and dermatitis exfoliativa, but this seems more likely a coincidental parallelism due to some other factor than DNP.
Because of the potential dangers of overdosage in uncontrolled use, and indiscriminate labeling of the myriad weight-reduction preparations containing DNP, the drug was removed from the market by the FDA. in 1938 [Parascandola, J. supra], and in 1939 the state of California made it a felony to sell, dispense, administer, or prescribe DNP for human consumption [Horner, D. W. supra]. The intent of these laws was manifestly to prevent public misuse and overuse of DNP as a weight reducing agent.
DNP or related phenol-derivatives have been used as skin cosmetics or therapeutic compositions for treatment of skin irritations [U.S. Pat. No. 2,281,937; Japanese patents Nos. 46-9158 and 46-5837]. DNP and related phenols have also been suggested as active agents in insecticides [U.S. Pat. No. 2,166,121; 2,210,894; 2,210,929], and in rat control preparations (Italian Pat. No. 440144).
The effect of DNP on one form of animal tumor in vivo was briefly investigated in 1933, but without the associated nutritional regimen provided by the present invention [Emge, L. A. et al. Proc. Soc. Exp. Biol. Med. 31, 152 (1933)]. In Emge, sarcoma tumors in rats injected with DNP did not show any macroscopic changes in growth rate.
The present applicant found in a preliminary evaluative clinical trial with far-advanced human cancer patients having histologically-verified malignancies representing a wide range of cancer types (breast, colon, lung, prostate, larynx, lymphoma) that a significant rate and extent of reduction in tumor size occurred when DNP was administered in coordination with a calorically and compositionally defined nutritional regimen defined individually for each patient, according to the present invention. The team of professionally qualified biochemists and medical oncology specialists monitoring the patient status throughout this clinical evaluation reported an absence, throughout the treatment regimen, of any discernable toxic side effects.
The therapy system of the present invention substantially avoids several of the traditional problems and limitations of conventional mitoxin chemotherapy. Mitoxin chemotherapy characteristically acts by the indiscriminate destruction of mitotic cells in the body, both normal and malignant. Because of this indiscriminate destruction of normal dividing cells by mitoxin chemotherapy, a host of toxic and treatment-limiting side effects are experienced, including anemia, pronounced loss of cellular and humoral immune competence, decrease of blood platelets, gastrointestinal ulceration with vomiting and diarrhea, electrolyte imbalance, anorexia, loss of hair, abnormalities of the nervous system, kidney damage, skin rash, liver damage, abnormal heart beat, and damage to the lungs. The present method of metabolic chemotherapy, because it does not adversely affect normal dividing cells in the body, is strikingly free of all such toxic effects and therefore permits continued administration until potentially all of the malignant cells are destroyed.
Similarly, since the present method does not destroy blastogenic lymphocytes of the immune system as does mitoxin chemotherapy, the body's immune competence remains unaltered, thus avoiding the pronounced decrease in resistance to infectious disease usually seen in human patients undergoing chemotherapy, and maximally enhancing potential immunological cell-mediated and humoral attack on residual tumor cells.
Furthermore, the present invention substantially avoids the traditional mitoxin-chemotherapy problem of resistant malignant-cell variants arising by mutation during the course of cancer therapy. The uncoupling effects produced by DNP and other physiologically tolerable uncoupling agents of similar function do not generally depend upon the reaction with a specific functional protein (e.g., an enzyme) or upon the chemical structure of the uncoupling agent [See Heytler, P. G. "Uncouplers of Oxidative Phosphorylation" in Erecinski et al. (eds.) Inhibitors of Mitochondrial Functions Pergamon 1981, p. 203], unlike the situation in the case of the often-mutagenic mitoxin chemotherapeutical agents. Hence, it is unlikely that populations of cancer-cell mutants resistant to uncoupling agents will arise (e.g., by one-step mutations) in the course of treatment with the present method.
Additionally, since the present method does not require a cancer cell to be in the proliferative or dividing cycle in order to effect its lysis, the present method is fully and continuously effective against even those generally present clones of mitotically quiescent malignant cells which are entirely unaffected by the conventional mitoxin chemotherapeutical drug and therefore survive to produce continual tumor recurrences following the conventional mitoxin chemotherapeutical treatments.
A most significant advantage of the present method is the pronounced increase in O/P uncoupling effectiveness produced selectively in the cancer cells relative to that produced in normal cells by the same dosage of uncoupling agent, particularly by the most preferred (2,4-dinitrophenol) and preferred uncoupling agents of this invention. Since the uncoupling effectiveness of the classical O/P uncoupling agents [Heytler, P. G., 1981, supra]depends upon their lipid solubility [Hemker, H. C. supra], and since the lipid solubility increases very rapidly as the intracellular pH is lowered (i.e., acidity is increased) [Hemker, H. C. ibid], the relatively very low pH of the cancer cells resulting from the very high rate of lactate formation under the therapeutical conditions results in a pronounced selective increase in O/P uncoupling, and a commensurate decrease in net ATP production rate, in the cancer cells. Thus, for a given dosage level of UA, the uncoupling produced in the cancer cells may be selectively magnified up to several times that produced in the normal cells.